Project Summary Gangliosides, analogous to biological antennae in cells, are involved in critical roles across multiple biological processes (i.e. pathogen infection, cell-cell communication, inflammation). Neuroprotective and neurorestorative functions of gangliosides have been explored for their therapeutic application in Parkinson?s disease and other neurodegenerative diseases. Despite such important role of gangliosides, there is limited access of gangliosides. Majority of the current commercially available gangliosides are obtained from animal brains, which is likely a concern for future therapeutic applications. Such limited availability of gangliosides is further beset by the diversity based on different sugars and fatty acids. The technologies presented here cover an amalgamation of nearly a decade of research, where complex ganglioside is synthesized in a single reaction vessel followed by a simple purification technique. This biosynthetic tool opens the availability of critically needed gangliosides in research and support its therapeutic uses. In this fast track proposal, we will address some major challenges in transitioning this prototypic one-pot multienzyme (OPME) technology for large scale commercialization. First challenge is manufacturing of starting materials (glycosyltransferases, supplemental enzymes and lactosylsphingosine) to reduce the cost to manufacture gangliosides. Additional improvements of existing glycosyltransferases will further reduce the cost burden to manufacture gangliosides by improving catalytic efficiencies of the enzymes. Lastly, OPME scaleability will be explored for manufacturing the various gangliosides. A study will result in several commercial products, ranging from purified enzymes, miniaturized glycosylation kits and several key gangliosides with high commercial value. GM1 ganglioside has been reported to delay onset of Parkinson disease, and clinical trials will likely require animal-free gangliosides. A miniaturized kit will be provided as a tool for researchers to synthesize their own customized gangliosides. The glycosyltransferases and the OPME kits allow the broad scientific community to access these simple synthetic tools for other oligosaccharides, glycoconjugates, and glycoproteins. By expanding this OPME and lowering the cost of these products, additional information on the biological functions of gangliosides will be gained which will accelerate the development of ganglioside-based diagnostics and therapeutics. A positive feedback loop, where lower cost will increase purchase volume, which will feed additional studies and will lead to the further growth of the market for the glycosyltransferases, OPME kits, and highly pure, animal-free gangliosides.